A RATIONAL DESIGN APPROACH FOR GENOTYPING OF BOVINE EPHEMERAL FEVER VIRUS (BEFV) USING HIGH RESOLUTION MELTING (HRM) ANALYSIS

Genotyping of viral pathogenic strains is important for epidemiological, immunological, phylogenetic and ecological research. Sequencing is the most useful method for genotyping, but it is relatively time-consuming, expensive and laborious, when rapid diagnosis is required. In this study a combined high resolution melting (HRM) real-time PCR-based identification was developed, for the viral pathogen BEFV using a rational primer design. BEFV (order Mononegavirales, family Rhabdoviridea) affects livestock in Asia, Africa, The Middle-East and Australia, leading to considerable economic damage due to a sharp loss of milk and weight, abortions and export restrictions. In order to distinguish between isolates from different regions, the sequence of the envelope glycoprotein G from isolate groups of different geographic origins was scanned to identify segments that are similar within each group, but different between the groups. The GC% differences within and between groups in these regions were established, and the gene regions were ranked according to their differences. Based on this analysis, primers were designed that correspond to the margins of the most highly-ranked regions. By combined examination of 4 such primer pairs, differentiation between isolates from Israel, Turkey, South-Africa, Japan and Australia was performed. In the examined sequences, there was no correlation between geographic distance and melt point difference, but the HRM pattern was consistent with sequence-based phylogenetic analysis. The method developed herein is a general tool for designing HRM differentiation systems. The application of this method in local BEFV diagnostics enables differentiation between vaccinated and naturally-infected animals, and tracing the origin of introduced strains that are new to this region.